Review Article

Establishing a stem cell culture laboratory for clinical trials

Elíseo Joji Sekiya1 Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their
Andresa Forte2 isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs
Telma Ingrid Borges de Bellis Kühn2 and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel
Felipe Janz3 with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The
Sérgio Paulo Bydlowski3
rules for the implementation of cell therapy centers involve national and international standards of good
Adelson Alves4
manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and
scientific development. Here standards from the United States, the European Union and Brazil are analyzed.
1
Instituto de Ensino e Pesquisa São Lucas, Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for
São Paulo, SP, Brazil the preparation and supply of cultured cells for clinical studies are described. Development stages involved
2
CordCell Centro de Terapia Celular,
the planning and preparation of the project, the construction of the facility, standardization of laboratory
São Paulo, SP, Brazil
procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge
3
Laboratório de Genética e Hematologia
Molecular (LIM31), Faculdade de Medicina of research centers involved in the study of cells with the practical experience of blood therapy services that
da Universidade de São Paulo - FMUSP, manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to
São Paulo, SP, Brazil implement cell therapy centers.
4
Hemocentro São Lucas, São Paulo, SP, Brazil

Keywords: Stem cells; Tissue therapy; Cell culture techniques; Good manufacturing practices.

Introduction

At the beginning of the twentieth century, in vitro tissue culture methods were developed
to study the behavior of animal cells free of in vivo systemic variations. These cells could be
tested under stable conditions imposed in the experiment. Cell culture methods were limited to
applications that involved primary explants which at that time were the only source. In 1916,
enzymatic dissociation of tissue was demonstrated by Rous & Jones(1) and a methodology of
cell expansion became possible.
One hundred years after the discovery of these methods, researchers have changed
the focus of cell culture procedures. In addition to cell studies into viral behavior and
pharmacological agents, the current goal is ex vivo cell expansion. This approach will allow
cells to be used in clinical treatments, the goal of cell therapy.
Another important achievement was the isolation of the first human cell line from a uterine
cervical tumor (HeLa). This gave rise to experimental studies in cancer and other diseases, such
as poliomyelitis(2). In this area, stem cells played a prominent role, due to the transdifferentiation,
self-renewal and paracrine effects that they display in some tissues and systems.
However, in many cases appropriate stem cells/progenitors are not available in sufficient
quantities to restore organs and damaged tissues. To address this issue, numerous studies have
developed ways to expand a limited number of cells in order to acquire a sufficient quantity
for therapeutic use. Advancements from these studies might contribute to the development of
Conflict-of-interest disclosure:
Andresa Forte works for CordCell Centro de regenerative therapies(3).
Terapia Celular, São Paulo, SP, Brazil The complexity of these new technologies makes clinical applications difficult(4). These
technologies require adequate facilities, qualified staff, techniques to evaluate cell quality
Submitted: 2/7/2012
Accepted: 4/7/2012
and well defined protocols for cell production. The rules to establish adequacy in these areas
are at an advanced stage; they are based on national and international standards of good
Corresponding author: manufacturing practices (GMP). Generally, evaluation points have to be planned and require
Elíseo Joji Sekiya major investments from cell therapy centers(5).
Rua Itatiara, 153
01242-000 São Paulo, SP, Brazil
Phone: 55 11 3660-6000 Standards and legislation
iep@iepsaolucas.com.br
Major breakthroughs in biotechnology, biology and medicine have promoted new
www.rbhh.org or www.scielo.br/rbhh therapeutic strategies in cell therapy, including stem cell transplantation. Biologically
privileged stem cells have been used for research and for clinical applications. However,
DOI: 10.5581/1516-8484.20120057 legislation and GMP standards are not uniform throughout the world.

236 Rev Bras Hematol Hemoter. 2012;34(3):236-41


Several standards have been published for the structuring
of cell processing centers. The Food and Drug Administration
(FDA) is responsible to ensure the safety of biological products
used in humans in the United States(6) and regulates the processing
of ex vivo cell products(7). These processes are divided into two
categories: minimally manipulated and effectively manipulated.
Minimally manipulated products are those that were only
submitted to procedures that did not alter the basic features and
function of cells. These procedures include cryopreservation
and cell thawing, density gradient separation, washing and the
dilution of cell products before reinfusion, cell selection and cell
depletion. Effectively manipulated products are those submitted
to procedures that might have altered the biological features of
cells. This includes ex vivo expansion and genetic modification.
The FDA uses a risk-based approach to regulate cell products in
both categories. A facility that supplies minimally manipulated
products should follow the current Good Tissue Practice (cGTP)
guidelines as defined in the “Code of Federal Regulations” (CFR)
title 21, part 1271(8). A manufacturing laboratory that provides
effectively manipulated cells should follow the cGMP guidelines
defined by the CFR title 21, part 211(9), in addition to applicable
items in the category of minimally manipulated products.
In Europe, countries that are members of the European
Union (EU) define cell-based medicinal products (CBMP) as any
viable cells (including stem cells) used in clinical treatment(10).
The European Medicine Agency (EMEA), established in London
by Regulatory Council nº 2309/93 of 22 July 1993, is responsible
for coordinating existing scientific resources for country
members that seek assessment and supervision in the production
of medicinal products for human and veterinary use(11-13).
CBMPs are heterogeneous in origin, cell type and complexity.
A living organism has stem cells, progenitor cells (with a
higher degree of differentiation) and terminally differentiated
cells (with a defined physiological function). In addition, cells
can be genetically modified, used alone or in association with
biomolecules or chemical substances, or combined with structural
materials (combined products)(14-17).
The European and American legislation on GMP defines
qualities that are required for cells used in the treatment of
patients. These qualities include donor suitability, cell product
traceability, follow-up of the donor and the patient over time
and quality control systems with documentation that must be
maintained by the handler(18-20).
Many institutions have recently built or are interested in
building manufacturing facilities to support clinical research
in Brazil and around the world. However, for facilities to be
classified “GMP compliant”, it is not sufficient just to build the
facility according to the prevailing building standards (CFR part
211). These standards include eleven sub-parts and the building
structure is only one item(21).
For laboratories designed to prepare effectively manipulated
cells, the standards take into account the following critical points:
general planning, organization and human resources, facilities
and edifications, equipment, component controls, containers,
manufacturing and process control, packing and labeling, storage
and distribution, laboratory controls, records and reports, returned
products and remaining products.
Rev Bras Hematol Hemoter. 2012;34(3):236-41
Establishing a stem cell culture laboratory for clinical trials
In Brazil, the Regulatory agency, ANVISA, recently
published Resolution nº 9 of March 14th, 2011(22). This resolution
provides guidelines for the operation of cell technology centers
(CTCs) that prepare human cells and derivatives for the purpose
of clinical research and therapy. The guidelines set the minimum
requirements for these CTCs.
The Brazilian legislation has two classifications for CTC
facilities:
• CTC Type 1: An establishment that only performs activities
with human adult cells that are autologous, fresh or
cryopreserved without cultivation and have been only
minimally manipulated for use in clinical research or
therapy or both.
• CTC Type 2: An establishment that performs activities
with human stem cells, adult or embryonic, autologous
or allogeneic, fresh or cryopreserved, with or without
cultivation, and with or without extensive manipulation for
use in clinical research or therapy or both.
The project must adhere to the mandates in Resolution
RDC/ANVISA nº 50 of February 21st 2002 (23), which regulates
the planning, programming, elaboration and assessment of
physical projects in healthcare facilities. The structure should
be used and accessed exclusively for the approved purpose. To
avoid cross-contamination, the flow of raw materials, biological
materials, professionals and waste should be independent; in
addition, allowances should be made for thorough cleaning and
maintenance of equipment and tools.
The deployment process: experiences of a Brazilian
cell technology center
Planning and project elaboration
Researchers in basic research with proficiency in cell cultivation
were consulted to create a project that would be developed in a CTC.
Despite great technical contributions in laboratory processes, to
fulfill the aim of manipulating cells for clinical applications, it was
necessary to consult a professional with extensive knowledge of
practical and management aspects of project design.
Thus, a team was created that had extensive experience in
managing the scientific, technical, and administrative aspects of
medical services in the area of hemotherapy and bone marrow
transplantation. The objective of the transfusion treatment was to
provide blood cell components that would restore cellular elements
in a patient with a deficiency. The transfusion would be derived
from total blood either from the patient or from other donors(24).
In bone marrow transplantation, the hemotherapy would be
performed with bone marrow, peripheral blood or umbilical cord
blood(25-26). The hemotherapy would involve hematopoietic stem
cell harvesting, processing, cryopreservation and infusion after
chemotherapeutic conditioning(27).
Because Brazil lacked specific national standards, the final
CTC project design was based on a review of all legislation
available at that time that regulated activities in this area. Thus, the
project was compatible with requirements in the Brazilian scenario.
237
Sekiya EJ, Forte A, Kühn TI, Janz F, Bydlowski SP, Alves A
Physical structure
The CTC was designed to conduct all procedures related to
stem cell preparation. These included harvesting, cell processing,
storage, quality control of cells, disposal, release for use and
transport(28-30).
According to the classification of clean room types
established in the national standards RDC 9 of 2011 (cited above),
centers with existing stem cell processing labs were considered
CTC type 1. Thus, these were considered establishments that
only performed activities with human autologous, fresh or
cryopreserved adult stem cells, without cultivation for use in
clinical research and/or therapy.
The purpose of the current project was to achieve a laboratory
with type 2 classification.
According to international standards, the classification of
air quality in each area is defined by the nature of cell products
that are manipulated in the laboratory. Currently, there are two
accepted classifications in the world: the air classification of
the US Federal Government Standard and the International
Organization for Standardization (ISO)(31). Table 1 illustrates the
differences between these classifications.
Table 1 - Air classification standards: comparison between US and ISO
guidelines on viable count action levels(32)
Clean room classification - Number Active air
ISO
US guidelines of particles action levels*
(Particles 0.5 µm/ft3) designation
> 0.5 µm/m3 (CFU viable/m3)
100 5 3,520
1000 6 35,200
10,000 7 352,000
100,000 8 3,520,000
*Plates of microbiological culture with diameter of 90 mm; CFU/4 h
ISO = International Organization for Standardization; CFU = Colony Forming Unit
When cell processing is conducted in an open system, it
should be performed in a biological cabinet that conforms to
ISO-5 specifications (US Class 100) placed in an environment
classified as ISO-7 (US Class 10,000). When product
manipulation is conducted in a closed system, with minimal
manipulation and aseptic validation, it may be performed in a
non-classified environment, but with a controlled air system. Cell
culture laboratories must have positive pressure relative to the
surrounding areas to avoid the inflow of contaminated air.
To comply with international standards related to the
physical structure of a clean room, it was necessary to select
engineering companies with specific experience in building and
installing GMP facilities. Because no national legislation existed
to regulate this area, company selection was a long, difficult
process, due to discrepancies in the information obtained. Thus,
the company selected proposed a project that fully complied with
US regulations utilizing the existing structure.
Considering that this was the only structure of its kind in
Brazil, the next task was to set up an information and experience
exchange network with international sites of excellence in cell
processing for human use. For example, correspondence was
238
established with the MD Anderson Cancer Center (Houston,
Texas, USA), the New York Blood Center (New York, USA) and
the Duke University Medical Center (Durham, USA).
After selecting the engineering company, the next
step was to acquire the proper equipment to purify the
room air. The equipment had to generate an environment
with the recommended number and quality of air particles.
The accepted guidelines mandated inert particles in this
environment, with no detectable microbiological activity.
Therefore, we acquired equipment that was available and
approved for the project. This was the most time consuming
part of the project, because most equipment was imported,
and the deadlines could not always be met. The location of
equipment in controlled environments also had to follow
specific standards that were included in the project.
To suit international standards, the laboratory was built
with one class 100,000 (ISO 8) clean room and two class
10,000 (ISO 7) clean rooms. Access from the external to the
internal environments required passage through two separate
rooms. One of these rooms was for washing the hands and
the other was for putting on clean laboratory attire. To access
the class 10,000 environments, we also built a specific area
to prevent cross-contamination. In each access room, stickers
were fixed to the floor to retain particles that could be dragged
between rooms.
The air treatment system and air purification equipment
was designed and installed in a protected environment. The
environment was turned into a clean room with HEPA filters
1 and air compressors to substitute environmental air. Particle
7 count sensors were also installed at strategic points to provide
10 continuous air quality monitoring and automatic activation of an
100 insufflation and aspiration air system. Humidity sensors and air
pressure monitors were also installed.
Dedicated equipment was installed inside the class 100,000
laboratory. The water treatment system (Ultrapurification Water
System, Milli-Q) produced ultrapure water with pass-through
ultraviolet radiation. Precision scales were also included as were
a freezer to store reagents, refrigerators, biological safety cabinets
(Class 100; ISO-5), a refrigerated centrifuge and an anaerobic
CO2 cabinet.
Once the structure was ready and the equipment was properly
installed and tested, the qualifying phase of the qualitative and
quantitative control of laboratory air was started. To ensure the
reliability of results, organizations that were not connected to
the engineering company that installed the air treatment system
were hired. One company was engaged to check and certify the
sterile conditions of the laboratory environment, including the
control of air particle density and the air outflow system and other
companies to assess the microbiological control.
Standardization of laboratory procedures
After concluding the structural evaluation, another
fundamental phase of cGMP structure maintenance was
begun(33-35). This included the standardization of methods,
procedures and materials to perform the activities and maintain
hygiene in the controlled environments(36).
Rev Bras Hematol Hemoter. 2012;34(3):236-41

Cross-contamination
Asepsis is the most important requirement that distinguishes
cell culture protocols for materials to be used in humans from
laboratory techniques(37-40). However, establishing sterile areas
requires high investment. The areas must maintain strict hygienic
controls (dirt removal with special products), restricted access,
an established flow of authorized individuals, and proper attire
dedicated for use in this area. These structures should also
incorporate a unidirectional flow of raw materials, components,
products, employees, and discharges to ensure maximum
segregation and minimum intersection.
Cleaning
The sanitization of clean rooms requires the use of suitable
pharmaceutically pure water or water purification systems that meet
USP standards. Solutions for cleaning and sanitizing equipment
within these facilities must be produced with suitably pure water.
Movement of individuals
In designing the physical area of the clean rooms, it was
important to scale the room sizes to the number of employees
assigned in order to control room cleanliness, the time required for
each shift, and what types of culture would be developed. To ensure
site cleanliness, norms were established to control the number of
people allowed inside and the flow of people. This design was also
useful to determine the number of biological safety cabinets and
the types of equipment. Another important issue was to avoid a
large number of professionals and equipment operating at any one
time. This decreases air circulation, and consequently, minimizes
the risk of contamination by microbial particles.
Quality control of the system and standardization of
protocols
To implement the laboratory routine, it was important
to map processes, identify and delineate critical procedures
and establish procedures to monitor the products and expected
results. In monitoring products for clinical use (in humans), it
is essential to have laboratory tests for the quality control of
cultured cells. Flow cytometry was performed to characterize the
cells and cytogenetic analysis was used to monitor chromosomal
alterations. Tests for in vitro cell differentiation and sterility,
among others, were also standardized.
Parameters considered critical for proper procedures
were analyzed and validated. For example, the transportation
time of biological materials, cultivation protocols, laboratory
garments, aseptic procedures, materials, reagents etc. Results
were presented and discussed in a scientific committee to gain
institutional approval.
Traceability was established for the entire procedure,
materials used, and professionals employed through standardized
registration forms and a system for packaging, storage, and
distribution. A program for waste management was established
according to current national standards.
Rev Bras Hematol Hemoter. 2012;34(3):236-41
Establishing a stem cell culture laboratory for clinical trials
Discussion
An important future direction of medicine is the development
of cell-based regenerative medicine. As, in many cases, humans
cannot regenerate damaged tissue, there is a clear need for stem
cell isolation and ex vivo expansion(41,42). These activities require
an appropriate laboratory environment to ensure adequate safety
during cell processing to eliminate risk of mortality(43).
The new Brazilian legislation describes the minimum
environmental conditions required for centers that offer cell
technology. However, this is only the first step in regulating the
complex processes involved in cultivated stem cell delivery.
The main difficulties in deploying a Brazilian CTC were related
to discrepant information that arose due to the lack of regulations in
force in Brazil. Some difficulties were addressed by the selection of
specialized engineering companies. Building proposals were presented
to the team responsible for the project. Technical issues ranged from
maintaining air quality to problems related to coatings on equipment.
International exchange allowed us to assess similar laboratories; this
provided crucial information for decision making on key issues.
The GMP guidelines for cell harvesting, processing,
cryopreservation, testing, distribution, and the use of blood
products enabled the implementation of a safe production system
for cultured cells. Another important consideration was quality
control, which must be applied not only to the final product (the
cells), but also to each procedure involved.
Beyond the technology, the most important goal is patient
safety. The team management experience allowed us to achieve
the project and implement safe procedures for processing stem
cells from various sources for use in studies involving humans.
Conclusion
Cell therapy is a medical area on the rise. This has increased
the demand for CTCs that can supply stem cells suitable for
clinical protocols. In Brazil, the legal framework for regulating
these structures was recently established. Previously, CTCs were
rarely prepared to meet all the requirements for the production
of cells for use in humans. The solution presented here is a
practical response to the needs of basic research specialties
that deal with cells. We described the practical management
concerns involved in establishing an institution that must also
meet demands for constant improvement and exchanged ideas
with other institutions involved in this process. We demonstrated
that a theoretical knowledge of site requirements combined with
practical experience in hemotherapy services provides the best
synergy for meeting the demands of establishing a viable CTC.
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